Apoptosis Induced by Differentiation or Serum Deprivation in an Immortalized Central Nervous System Neuronal Cell Line

Abstract: To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1β converting enzyme (ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 T^ts-immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-x_L, and this could be reversed by bcl-x_S.Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-X_L-expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-X_L. Furthermore, studies involving expression of BCL-2 and BCL-X_L alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cellsoccurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-X_L can prolong the survival of differentiated neuronal cells.     

Biochemical and Cellular Properties of Three Immortalized Schwann Cell Lines Expressing Different Levels of the Myelin-Associated Glycoprotein

Abstract: Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle-shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P₀ glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin-related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin-related components.     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidopteran Cell Lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33℃ and 35℃ was carried out by shifting the culture ...     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Activation of Mitogen-Activated Protein Kinase by Epidermal Growth Factor in Hippocampal Neurons and Neuronal Cell Lines

Abstract: Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.     

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

Rapid Determination of Norepinephrine, Dopamine, Serotonin, Their Precursor Amino Acids, and Related Metabolites in Discrete Brain Areas of Mice Within Ten Minutes by HPLC with Electrochemical Detection

A rapid and simple chromatographic procedure using HPLC with electrochemical detection is described for simultaneous determination of the substrates from precursor amino acids to metabolites related to synthesis and metabolism of three monoamine neurotransmitters--norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin)--in discrete brain areas of the mouse. Under the present instrumental and mobile phase conditions, the procedure permits simultaneous determination of three monoamines (NE, DA, and 5-HT), two precursor amino acids (tyrosine and tryptophan), and four respective metabolites (3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid) within 10 min in one chromatographic run. By varying column temperature, this procedure also permits simultaneous determination of 10-14 monoamine-related substrates including the nine substrates described above within 15-21 min. The validity of the present procedure is demonstrated by analyzing the effect of an alpha 2-adrenergic agonist (clonidine) and an alpha 2-antagonist (yohimbine) in mouse hypothalamus.     

Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.     

Neuronal and Glial Properties Coexist in a Novel Mouse CNS Immortalized Cell Line

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.     

Cytotoxicity of Nephrotoxic Fungal Toxins to Kidney-derived LLC-PK1 and OK Cell Lines

The nephrotoxic fungal toxins ochratoxin A (OA), ochratoxin B (OB) and citrinin (CIT) are natural contaminants of foods and feeds. While cytotoxicity assays have proven useful for establishing relative toxicity and structure–function relationships within groups of fungal toxins, a drawback of in vitro bioassays is their susceptibility to variation depending on endpoint, target cell, and dosing strategy. These variables were explored for OA, OB, CIT using two continuous kidney cell lines (LLC-PK₁ and OK) and four cytotoxicity assay endpoints. The nephrotoxic antibiotic gentamicin was used as a positive control for cytotoxicity throughout. In general, fungal toxin-induced cytotoxicity was more pronounced in LLC-PK₁ cultures using mitochondrial dehydrogenase inhibition (MTT assay) as the endpoint. Altered dosing strategy, but not seeding density, consistently influenced cytotoxicity: CIT was more toxic to cells when added at the time of seeding, whereas OA was more toxic when added 24 h after cultures were seeded. Toxicity rankings for the fungal toxins were consistent with in vivo studies and were, in order of most to least toxic, OA>OB>CIT. The data indicate that LLC-PK₁ and OK cells compare favorably to existing models in terms of sensitivity to nephrotoxic fungal toxins, but also that relatively minor changes in assay protocols can affect the cytotoxicity of individual toxins and comparative toxicity within a group of toxins.     

Targeting and Processing of Pro-Opiomelanocortin in Neuronal Cell Lines

Pro-opiomelanocortin (POMC) is the precursor to several pituitary hormones including adrenocorticotropic hormone and beta-endorphin (beta-END). POMC is also expressed in the brain, predominantly in discrete neuronal cell populations of the hypothalamus. In the pituitary and brain, POMC undergoes tissue-specific proteolysis to release different bioactive peptides. POMC processing in neuronal cell lines was studied after infection of PC12 and Neuro2A cells with a recombinant retrovirus carrying the porcine POMC cDNA. Our results indicate that both cell lines synthesize and target POMC to the regulated secretory pathway. Only the Neuro2A cells, however, can achieve proteolytic processing of POMC. Chromatographic and immunological characterization of the POMC-related material showed that beta-lipotropin (beta-LPH) and nonacetylated beta-END(1-31) are major maturation products of POMC in these cells. Release of both beta-LPH and beta-END(1-31) from infected Neuro2A cells can be stimulated by secretagogues in a calcium-dependent manner. Taken together, our results suggest that the cellular machinery of Neuro2A cells can recognize a foreign prohormone, target it to neurosecretory vesicles, process it into biologically active peptides, and secrete it in a manner characteristic to peptidergic neurons.     

PC-1基因在前列腺癌细胞系LNCaP和C4-2不同细胞周期的表达

目的:检测PC-1基因在前列腺癌细胞周期中各时间点的表达变化。方法:用200 ng/mL诺可唑(nocoda-zole)处理前列腺癌细胞系LNCaP和C4-2,16 h后使细胞处于G2/M期,在不同时间点收获细胞,分别进行流式分析和Western印迹,检测PC-1基因的表达。结果:流式分析和Western印迹结果显示,在G2/M期,LNCaP和C4-2前列腺癌细胞系中PC-1基因高表达。结论:PC-1基因的表达与前列腺癌细胞的细胞周期有关,提示PC-1可能在细胞周期调控中发挥作用。     

Immunogenicity of Ehrlichia ruminantium Grown in Tick Cell Lines

Ehrlichia (previously Cowdria) ruminantium, the pathogen which causes heartwater in domestic and wild ruminants, can now be propagated in cell lines from one vector (Amblyomma variegatum) and five non-vector (Ixodes scapularis, I. ricinus, Boophilus decoloratus, B. microplus and Rhipicephalus appendiculatus) tick species. E. ruminantium isolates from West and South Africa and the Caribbean vary in their cell line preference, growth patterns and immunogenic capability. In laboratory trials, certain combinations of tick cell line and E. ruminantium isolate were highly immunogenic in sheep. These trial vaccines were grown under specific in vitro conditions and administered as a single intravenous dose of freshly harvested whole, live culture. Following immunisation and subsequent exposure to virulent E. ruminantium, protected sheep showed no clinical response and a range of serological responses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Notchl信号蛋白在卵巢癌细胞系中的表达

目的：检测卵巢癌细胞系中Notchl信号蛋白的表达,为在卵巢癌细胞中对Notchl进行RNA干涉研究的体外实验筛选靶细胞。方法：采用逆转录聚合酶链反应（RT-PCR）法及Westernblotting对卵巢癌细胞系SKOV3、HO-8910、HO-8910-PM和3AO中Notchl进行检测。结果：Notchl信号蛋白在4种卵巢癌细胞系中均有表达,其在HO-8910-PM中表现为高表达,在SKOV3和3AO中表现为中等表达,在HO-8910中表现为低表达。结论：HO-8910-PM适合作为靶细胞,进行Notchl信号蛋白的RNA干涉研究。     

施氏鲟不同组织来源细胞离体培养的初步研究

采用组织块移植培养技术,对来源于施氏鲟(Amursturgeon,Acipenser schrencki Brandt)肝脏、脾脏、肾脏、鳍条和心脏组织的细胞进行了原代培养,2～3天左右可见组织块周围有成纤维样细胞迁出,10天左右组织块周围形成单层细胞。对原代培养的单层细胞用胰蛋白酶-EDTA消化后传代培养,建立了可连续传代的施氏鲟肝脏、脾脏、肾脏、心脏组织细胞系。初步确立施氏鲟细胞培养的条件,培养基为MEME,培养温度为25℃,血清浓度为20%。对传代培养细胞以二甲基亚砜为保护剂在液氮冷冻保存,细胞复苏后可连续传代培养。施氏鲟细胞离体培养为开展鲟鱼病毒病和遗传资源保存研究提供了重要试验材料。     

水翁悬浮细胞系的建立及其悬浮培养的生长特性

建立了水翁悬浮细胞系,并对其悬浮培养的生长特性作了初步探讨。以水翁新生芽尖作为外植体,接种于添加有不同浓度和配比的生长调节物质及各种附加物的MS固体培养基中,诱导培养产生初代愈伤组织;挑选Ⅰ和Ⅱ型的愈伤组织进行继代培养改良,考察愈伤组织的生长状况和统计生长量来决定最佳继代培养基的配方和得到适合悬浮培养的愈伤组织;将以上得到的愈伤组织转接于最佳继代液体培养基中,于24±1℃,120r/min条件下振荡培养,筛选分散度好、较均匀、生长快、色浅透明的细胞作为种子传代,数次传代后得到性能良好的悬浮细胞系;以细胞生长量(鲜重)为指标,绘制了水翁悬浮细胞的生长曲线。研究表明:2.0mg/L的2,4-D的诱导率最高(92%,初代愈伤组织为Ⅰ型),Ⅱ型愈伤组织的最高诱导率为75%;最佳的继代培养基配方为MS 0.5mg/L 2,4-D 0.5mg/L 6-BA 1.0mg/L IAA 0.5mg/L IBA 0.5mg/L NAA 0.1mg/L KT 700mg/L LH,形成Ⅱ型愈伤组织的生长量可达3.28g/瓶(鲜重);液体继代培养3代后,可得到性能良好的悬浮细胞系;水翁悬浮细胞的生长曲线表明,最佳接种期为培养后的16~18d。     

Possible Role of Cyclic AMP in the Receptor-Mediated Regulation of Glycosyltransferase Activities in Neurotumor Cell Lines

Exposure of mouse neuroblastoma cell line N4TGl to opiates or [D-Ala2,D-Leu5] enkephalin produced a naloxone-reversible inhibition of cyclic AMP synthesis and prevented, in a concentration-dependent manner, the formation of both ganglioside GM2 (GalNAc-[NeuNAc]-Gal-Glc-ceramide) from GM3 (NeuNAc-Gal-Glc-ceramide) and ganglioside GM1 (Gal-GalNAc-[NeuNAc]-Gal-Glc-ceramide) from GM2 in cell-free extracts. In contrast, the receptor-mediated elevation of intracellular cyclic AMP levels by agents such as prostaglandin E1 (in the presence of isobutylmethylxanthine) or the addition of the cyclic AMP derivatives (dibutyryl cyclic AMP) markedly stimulated the activities of UDP-GalNAc:GM3,N-acetylgalactosaminyltransferase and UDP-Gal:GM2,galactosyltransferase. An overall increase in the synthesis of gangliosides more complex than GM3 was also observed in the mouse neuroblastoma x hamster brain explant hybrid cell line NCB-20 following elevation of cyclic AMP levels by treatment with serotonin and pargyline. The data presented support the hypothesis that cyclic AMP may have a role in the regulation of sialoglycosphingolipid biosynthesis.     

Comparison of Established Cell Lines at Different Passages by Karyotype and Comparative Genomic Hybridization

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.     

大鼠促甲状腺激素释放激素受体1基因的克隆及其在COS-7细胞系中的表达

目的:克隆大鼠促甲状腺激素释放激素受体1(TRH-R1)基因,构建其真核表达载体,并检测该基因在非洲绿猴肾细胞系COS-7中的表达。方法:应用RT-PCR方法,以大鼠脑源RNA为模板,扩增获得TRH-R1基因,定向克隆到pDsRed2-N1中,以LipofectAMINE 2000试剂转染pDsRed2-N1-TRH-R1表达载体至COS-7细胞系中进行瞬时表达。结果:测序结果表明,从大鼠脑源总RNA中克隆到正确的TRH-R1基因全长编码序列;显微照相观察到所构建的TRH-R1表达载体质粒在COS-7细胞系中获得有效表达。结论:大鼠TRH-R1基因的克隆、真核表达载体的构建及在COS-7细胞系中表达获得成功,为进一步研究其功能奠定了基础。